This invention relates to a use of known nucleases mixed in solution to reduce wrinkles and discoloration in humans.
In the double-helical structure of DNA, each strand of DNA consists of a backbone of phosphates alternating with ribose or deoxyribose groups. To each ribose or deoxyribose group is attached a base such as adenine or guanine. The two strands of the double helix are held together by hydrogen bonding, i.e., a hydrogen proton on a base attached to one strand is attracted to an electron pair of a corresponding base on the other strand. This attraction is analogous to two ropes, each of which has a series of magnets tied to it; the ropes can be pulled apart temporarily without tearing each magnet off of its rope, and the ropes can subsequently become re-associated via the attraction of the magnets. The two strands of a DNA molecule must be temporarily disassociated from each other in order for vital processes (including replication of DNA to allow cell reproduction, as well as transcription of RNA) to take place.
The process whereby two strands of DNA become hydrogen-bonded to each other is referred to as xe2x80x9cannealing.xe2x80x9d Because of the nature of annealing and hydrogen bonding, two strands of DNA that are not exactly complementary can be attracted to each other and will anneal with a substantial degree of stability under normal (e.g. physiological) conditions. In other words, if two strands of DNA inside a cell have similar but not identical sequences of bases, they can be attracted to each other and anneal. This can lead to various types of DNA recombination. For example, thymine residues can flex somewhat and form hydrogen bonds with guanine residues, even though thymine usually pairs up with adenine. Under in vitro conditions using Southern or Northern blot hybridization, two strands of DNA or RNA can xe2x80x9chybridizexe2x80x9d (become annealed to each other, even though they are not perfectly complementary) with as little as 55% homology. Strands with 80% or greater homology can remain attached to each other at temperatures well in excess of 37xc2x0 C., the normal temperature inside most cells in the human body.
In eukaryotic cells (i.e., cells that have nuclei, such as plant or animal cells) the chromosomes have substantial numbers of repetitive sequences. The highly repetitive sequences are often called the xe2x80x9cfast componentsxe2x80x9d of the genome, since they reassociate quickly in vitro after they have been denatured (separated) by means such as boiling. These repetitive sequences are not fully understood; it has been hypothesized that they may be involved in genetic recombination or in regulating gene expression.
Eukaryotic genes also have xe2x80x9cintronsxe2x80x9d. These are sequences of DNA in the chromosomal genes which are transcribed into sequences of messenger RNA (mRNA) that are excised, in the cytoplasm, from the primary mRNA transcript before the edited mRNA is translated into protein. Thus, introns are non-translated DNA segments within a gene. Introns (as well as edited mRNA) are eventually digested (i.e., broken down into their building blocks which are called nucleotides, each of which contains a phosphate group, a ribose ring, and a base) in the lysosomes in the cytoplasm, primarily by enzymes called xe2x80x9cnucleasesxe2x80x9d. However, at least some introns either diffuse back into the nucleus or are actively transported into it before they are digested into nucleotides. This is evidenced by the fact that viral nucleic acids, which must pass through the cytoplasm of a cell, quite frequently reach the nuclei of eukaryotic cells.
A nuclease which preferentially digests RNA (i.e., it digests RNA without digesting DNA at a comparable rate) is called an RNase (also capitalized as RNAse); conversely, a nuclease which digests DNA more rapidly than RNA is called a DNase or DNAse. Non-specific nucleases which digest either DNA or RNA at roughly comparable rates are simply called nucleases. A nuclease that digests a DNA or RNA strand at an internal position is an endonuclease. And, a nuclease that digests a DNA or RNA strand on a terminal end is an endonuclease. As used herein, the term xe2x80x9cnucleasexe2x80x9d includes all five categories of enzymes and combinations of the enzymes.
The terms xe2x80x9cpolynucleotidexe2x80x9d and xe2x80x9coligonucleotidexe2x80x9d are used to describe strands of nucleic acids having a sufficient number of bases to anneal with substantial avidity to a strand of DNA or RNA; those terms do not apply to single nucleotides, dinucleotides, or other strands too short to bind with any substantial avidity to a long strand of DNA or RNA having a complementary sequence. Avidity refers not just to the attraction between hydrogen-bonding molecules, but also to their ability to remain coupled together over a prolonged span of time.
As used herein, xe2x80x9cnon-chromosomal nucleic acidsxe2x80x9d refers to nucleic acid strands which are inside a living cell, but which are not bonded to the chromosome by a phosphate-sugar bond (i.e., they are not part of either primary strand in a chromosome; however, they might be hydrogen-bonded to one of the primary strands). Non-chromosomal nucleic acids include mRNA, transfer RNA, ribosomal RNA, and introns which have been removed from mRNA. It also includes any other strands of DNA or RNA which are in a living cell but which are not incorporated into a chromosome; this includes DNA or RNA from a virus which has infected a cell, as well as oligonucleotides or polynucleotides generated during digestion or as a result of cell death and lysis, which have been taken up by cells. Once inside the cell, the strands usually are digested further, into single nucleotides that function as metabolites for the cell.
The term xe2x80x9chuman tissue skin cellsxe2x80x9d refers to integument or skin cells in a human being and includes the epidermis and dermis layers of skin. The dermis includes the reticular and papillary layers, with the reticular layer including yellow elastic fibers and white tissue.
It has been hypothesized by others that wrinkles are caused by a breakdown in the connective tissue in the skin, typically yellow elastic fibers in the reticular layer of the dermis atrophy. Alternatively, the connective tissue may break down because excess protein produced inside the cells causes the cells to expand, which in turn causes the cells to wrinkle. It is believed that the excess protein that disrupts the connective tissue is produced as a result of oligonucleotides and polynucleotides annealing to the skin cell""s chromosome. This causes the regulatory mechanisms on the chromosome to function improperly, and thus proteins are over produced and disproportionately produced. By treating human skin cells with nucleases, protein levels return to close to their original level. Thus, the process either allows the connective tissue proteins to be produced at levels to maintain the skin integrity or decreases the skin cell size or both to reduce wrinkles.
To the best of the Applicant""s knowledge, no one has demonstrated an effective method for modifying the entry of non-chromosomal nucleic acids into eukaryotic cells, specifically a method of controlling gene expression in the skin cells, aside from caloric restriction or phlebotomy. Similarly, no one has previously suggested that oligonucleotides and polynucleotides which are taken into skin cells might interfere with the proper functioning of the genes. Finally, no one has suggested that preventing the over-production of protein will prevent the formation of wrinkles and reduce discoloration of the skin.
This invention involves a method of treating human skin with a solution containing nuclease so that the treatment results in the reduction of discoloration and wrinkles in human skin Specifically, the treatment involves altering the gene expression in human skin cells by means of contacting the skin cells with an effective amount of a nuclease solution containing an exogenous nuclease (an enzyme capable of degrading extra-cellular DNA and/or RNA). The nuclease in the nuclease solution will degrade extra-cellular nucleic acids into nucleotides or oligonucleotides which are too short to have substantial avidity for chromosomal DNA. This in turn will prevent oligonucleotides and polynucleotides from binding to the chromosome in human tissue cells, which prevents the over-production of protein and improper production of protein by individual cells. By altering the production of protein, wrinkles are reduced.
The nuclease solution is applied directly onto the area of the human body where it is desired to reduce wrinkles and discoloration spots, also known as xe2x80x9cage spotsxe2x80x9d. The solution may be applied one time or more than one time depending on the size and frequency of the wrinkles and discoloration spots.
The nuclease solution is formed by mixing together water, nuclease co-factor, and at least one nuclease. The nuclease is present in a concentration sufficient to digest at least one molecule of either DNA or RNA.